Primary structure of the alpha-subunit of vacuolar-type Na(+)-ATPase in Enterococcus hirae. Amplification of a 1000-bp fragment by polymerase chain reaction.

A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by the polymerase chain reaction method, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na(+)-ATPase alpha-subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of Na(+)-ATPase alpha-subunit is highly homologous to the corresponding sequences of large (alpha) subunits of vacuolar (archaebacterial) type H(+)-ATPases, supporting our proposal [Kakinuma, Y. and Igarashi, K. (1990) FEBS Lett. 271, 97-101] that the Na(+)-ATPase of this organism belongs to the vacuolar-type ATPase.     

Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone.

It has been reported that Escherichia coli is able to grow in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) when ATP is produced by glycolysis (N. Kinoshita et al., J. Bacteriol. 160:1074-1077, 1984). We investigated the effect of CCCP on the osmotic adaptation of E. coli growing with glucose. When E. coli growing in rich medium containing CCCP was transferred to medium containing sucrose, its growth stopped for a while and then started again. This lag time was negligible in the absence of CCCP. The same results were obtained when the osmolarity was increased by N-methylglucamine-maleic acid. In addition to adapting itself to the hyperosmotic rich medium, E. coli adapted itself to hyperosmolarity in a minimal medium containing CCCP, again with a lag time. Hyperosmotic shock decreased the internal level of potassium ion rather than causing the accumulation of external potassium ion in the presence of CCCP. The internal amount of glutamic acid increased in cells growing in hyperosmotic medium in the presence and absence of CCCP. Large elevations in levels of other amino acids were not observed in the cells adapted to hyperosmolarity. Trehalose was detected only in hyperosmosis-stressed cells in the presence and absence of CCCP. These results suggest that E. coli can adapt to changes in the environmental osmolarity with a negligible accumulation of osmolytes from the external milieu but that the accumulation may promote the adaptation.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Quantitative determination of partially methylated alditol acetate of amino sugar by gas chromatography-mass spectrometry

Using doubly labeled N-acetyllactosamine. Hakomori's procedures to prepare partially methylated alditol acetates and their subsequent analysis by gas chromatography-mass spectrometry were followed from a quantitative aspect. It was found that both galactose and glucosamine were nearly quantitatively converted to PMAAs. Preferential loss of PMAA of glucosamine occurred on a column for gas chromatography when the amount of the PMAA injected onto a column was less than a certain level. Above this level, PMAAs of both galactose and glucosamine were eluted from the column with equal yields. However, in mass spectrometry with monitoring of total ions, the molar response factor of PMAA of glucosamine was found to be about 25% higher than that of PMAA of galactose. Based on these findings, methylation analysis of an oligosaccharide from Taka-amylase A composed of one glucosamine and five mannose residues could be carried out quantitatively.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining.

Neutralization tests were made on 4 types of dengue (DEN) virus and Japanese encephalitis (JE) virus by incubation of serially diluted antisera and constant amounts of the viruses and then focus assay of surviving virus infectivity with peroxidase-anti-peroxidase (PAP) staining. Neutralization reactions were virtually completed in 2 hr on incubation of serum-virus mixtures at 28 C. A straight regression line was obtained on a probit chart by plotting the focus reduction rates at various dilutions of a given serum against the logarithm of the serum dilution used in the test. The slopes of the probit regression lines for the neutralization for DEN types 1 and 3 were similar, but differed somewhat from those for DEN type 2 and type 4. The slope of the line for JE virus was quite different from those for DEN viruses. Using these relations, the fifty percent focus reduction titer (FR50) of neutralizing antibodies of a given serum could be estimated from the focus reduction rates at several dilutions of the test serum when the latter was between 25-75% of the value of the control.     

Polyamine prevention of inhibition of rat liver isoleucyl-tRNA formation by poly(G), poly(I) or ribosomes.

It is shown that rat liver isoleucyl-tRNA formation in the presence of Mg²⁺ is inhibited by poly(G), poly(I) or ribosomes and that this inhibition is prevented by polyamines. The inhibition is found to be noncompetitive with respect to tRNA.     

Yeast RNA polymerase I: A eukaryotic zinc metalloenzyme

Microwave excitation spectrometry and metal binding inhibition studies show that zinc is a catlytically essential component of the highly purified RNA polymerase I from yeast, the first eukaryotic RNA polymerase I available in quantities sufficient for such studies. It contains 2.4 g-atom of zinc based on a molecular weight of 6.5 × 10⁵ (8). Copper, iron, manganese and magnesium are absent, i.e., below the limits of detection, 10^?13 to 10^?14 g-atoms. A number of derivatives of 1,10-phenanthroline reversibly inhibit the polymerase catalyzed reaction, apparently by forming a ternary polymerase·Zn·OP complex while the nonchelating isomer, 1,7-phenanthroline, is ineffective.     

Interaction of antimannan with glycopeptides

Taka amylase A glycopeptide (TA-GP) strongly inhibited the interaction of antimannan (antibodies directed towards mannan from $\text{Saccharomyces cerevisiae}$) with yeast mannan, whereas ovalbumin glycopeptide (OA-GP) did so only poorly. We inferred that this is due to the strong reactivity of antimannan with terminal trimannosides composed of Manα1→2Man or Manα1→3Man linkages which occur in mannan and TA-GP. In contrast, TA-GP and OA-GP were nearly equally reactive with concanavalin A having the ability to interact with terminal mannose and 2-0-mannose residues which occur abundantly in these glycopeptides. Thus, antimannan should be useful as a probe for characterizing glycoproteins from extracellular fluids or cellular membranes.